Evaluation of complement-dependent cytotoxicity using ATP measurement and C1q/C4b binding

One of the major issues for antibody treatment is enhancement of efficacy. Recent studies have highlighted the important role of effector functions in improvement of antibody therapy. Among effector functions, complement-dependent cytotoxicity (CDC), which induces cell lysis by a cascade of activation triggered by the binding of C1q subunits to the Fc regions of antibodies bound to the cell surface, is part of the mechanism of several antibody therapies. CDC can be modulated by either Fc isotype engineering or Fc genetic mutations or Fc glycosylation profile modifications. To evaluate the impact of such modifications on CDC, we describe a luminescence method based on ATP measurement to estimate tumor damaged cells and a flow cytometry method to evaluate the binding of C1q on the Fc region and the binding of C4b on cell surface. The luminescence method coupled with complement protein analysis by flow cytometry encompasses all needed methods to evaluate antibody ability to trigger CDC.